长链非编码RNA肌动蛋白纤维相关蛋白1-反义RNA1在口腔鳞状细胞癌中的表达及其相关功能

doi:10.7518/hxkq.2019.06.005

摘要/Abstract

摘要：

目的分析长链非编码RNA（lncRNA）肌动蛋白纤维相关蛋白1-反义RNA1（AFAP1-AS1）在口腔鳞状细胞癌（OSCC）中的表达及其对OSCC细胞生物行为学的影响,初步探讨其可能的作用机制。方法采用实时荧光定量聚合酶链反应（qRT-PCR）检测OSCC患者（55例）癌组织、癌旁正常黏膜组织及人口腔鳞状细胞癌SCC25、人正常口腔角质细胞株（NOK）细胞中lncRNA AFAP1-AS1的表达,分析AFAP1-AS1与OSCC患者病理特征的相关性,通过Kaplan-Meier生存曲线分析AFAP1-AS1与患者预后的关系。AFAP1-AS1 siRNA转染SCC25细胞,细胞计数（CCK-8）及Transwell实验分别检测细胞增殖、迁移和侵袭的变化,蛋白质印迹（Western blot）检测侵袭相关蛋白、肌动蛋白纤维相关蛋白1（AFAP1）及Rho GTP酶家族成员蛋白的表达情况,免疫荧光染色检测细胞骨架肌动蛋白微丝结构的变化。结果 OSCC组织中AFAP1-AS1的表达高于癌旁正常黏膜组织,SCC25细胞中AFAP1-AS1的表达高于NOK细胞（P<0.001）。AFAP1-AS1的表达与OSCC的分化程度、TNM分期及淋巴结转移密切相关（P<0.05）,AFAP1-AS1高表达患者的生存率低于AFAP1-AS1低表达者（P<0.05）。AFAP1-AS1 siRNA转染后AFAP1-AS1的表达水平下调,SCC25细胞的增殖、迁移和侵袭能力降低,AFAP1、RhoA、Rac2、Rab10、RhoGDI和Pfn1的表达上调,RhoC的表达下调,细胞骨架中应力纤维丝减少,肌动蛋白完整性丢失。结论 lncRNA AFAP1-AS1在OSCC组织及SCC25细胞中高表达,与OSCC的发生进展及预后相关。下调AFAP1-AS1能够抑制OSCC的增殖、迁移和侵袭能力,其可能是通过调控肌动蛋白纤维丝的完整性实现的。

关键词: 口腔鳞状细胞癌, 长链非编码RNA, 肌动蛋白纤维相关蛋白1-反义RNA1, 预后, 增殖, 侵袭

Abstract:

Objective To analyze the expression and clinical significance of long non-coding RNA (lncRNA) actin filament-associated protein 1-antisense RNA1 (AFAP1-AS1) in oral squamous cell carcinoma (OSCC) and its effect on the biobehavior of OSCC cells.Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA AFAP1-AS1 in the tumor tissue and matching adjacent normal tissue of OSCC patients (n=55), SCC25 cells, and normal oral keratinocyte lines (NOK) cells. The correlation between AFAP1-AS1 expression and the clinicopathological characteristics of OSCC patients was analyzed. The relationship between AFAP1-AS1 and prognosis was also studied with the Kaplan-Meier method. AFAP1-AS1 siRNA was transfected into the SCC25 cells. Cell counting kit-8 (CCK-8) and Transwell were used to detect cell proliferation, invasion, and migration. The expression of the invasion-associated protein, AFAP1, and Rho GTPase family members, was detected by Western blot. In addition, the immunofluorescence of the cytoskeletal actin filament was observed.Results The expression of AFAP1-AS1 was higher in the OSCC tissues than in the NOK cells, and the relative expression of AFAP1-AS1 was higher in the SCC25 cells than in the NOK cells (P<0.001). AFAP1-AS1 expression was associated with the degree of differentiation, TNM stage, lymphatic metastasis, and poor prognosis of OSCC (P<0.05). Patients with a high expression of AFAP1-AS1 had lower survival rates than those with a low expression of AFAP1-AS1 (P<0.05). After transfected by AFAP1-AS1 siRNA, the expression of AFAP1-AS1 was downregulated. The inhibition of AFAP1-AS1 expression consequently suppressed the proliferation, invasion, and migration of SCC25. Moreover, AFAP1-AS1 siRNA upregulated the expression levels of AFAP1, RhoA, Rac2, Rab10, RhoGDI, and Pfn1 but downregulated the expression of RhoC. Immunofluorescence showed that AFAP1-AS1 also reduced the formation of stress filaments in the cytoskeleton and affected the integrity of the actin filament.Conclusion The expression of AFAP1-AS1 was high in the OSCC tissues and SCC25 cells and is associated with the development and prognosis of OSCC. The knockdown of AFAP1-AS1 might inhibit the proliferation and invasion of OSCC cells by regulating the integrity of the actin filament.

Key words: oral squamous cell carcinoma, long non-coding RNA, actin filament-associated protein 1-antisense RNA1, prognosis, proliferation, invasion

中图分类号:

R739.8

耿玉东,王树斌,卢泰青,滕薇. 长链非编码RNA肌动蛋白纤维相关蛋白1-反义RNA1在口腔鳞状细胞癌中的表达及其相关功能[J]. 华西口腔医学杂志, 2019, 37(6): 594-601.

Geng Yudong,Wang Shubin,Lu Taiqing,Teng Wei. Expression and functions of long non-coding RNA actin filament-associated protein 1-antisense RNA1 in oral squamous cell carcinoma[J]. West China Journal of Stomatology, 2019, 37(6): 594-601.

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基因名称	序列（5′-3′）
GAPDH	上游：AGGCAACTAGGATGGTGTGG
	下游：TTGATTTTGGAGGGATCTCG
AFAP1-AS1	上游：ATCTTCGTTAGCACTGAGACTGG
	下游：AGAAAGAAGGTGGGAGTTAGGTG
si-AFAP1-AS1-1	CAUUAUUUUGCUAAUUCAACA
si-AFAP1-AS1-2	CGUUAUAUCUGAAAAAAAUCU
si-AFAP1-AS1-3	GAAGUUAGUACAUUAAACAAA
si-NC	CUGAAACAUACAGCAAUAAAG

临床病理特征		n	AFAP1-AS1低表达组	AFAP1-AS1高表达组	χ²值	P值
年龄/岁	≤60	31	18	13	2.289	0.130
	>60	24	9	15
性别	男	36	16	20	0.900	0.343
	女	19	11	8
肿瘤大小/cm	≤3	41	22	19	1.345	0.246
	>3	14	5	9
肿瘤部位	舌	24	14	10	1.582	0.663
	牙龈	15	6	9
	腭	4	2	2
	颊黏膜	12	5	7
分化程度	低分化	44	25	19	5.256	0.022
	中、高分化	11	2	9
TNM分期	Ⅰ、Ⅱ期	35	21	14	4.583	0.032
	Ⅲ、Ⅳ期	20	6	14
淋巴结转移	无	42	24	18	4.610	0.032
	有	13	3	10

组别	AFAP1-AS1的表达量	P值
空白对照组	1	-
si-NC	0.98±0.09	>0.05
si-AFAP1-AS1-1	0.58±0.06	<0.05
si-AFAP1-AS1-2	0.39±0.05	<0.05
si-AFAP1-AS1-3	0.72±0.06	<0.05